The present invention concerns molecules and methods for detection of specific agents in a medium.
Ribozymes are typically RNA molecules which have enzyme-like catalytic activities usually associated with cleavage, splicing or ligation of nucleic acid sequences. The typical substrates for ribozymes catalytic activities are RNA molecules, although ribozymes may catalyze reactions in which DNA molecules (or maybe even proteins) serve as substrates.
Ribozymes which are active intracellularly work in cis, catalyzing only a single turnover, and are usually self-modified during the reaction. However, ribozymes can be engineered to act in trans, in a truly catalytic manner, with a turnover greater than one and without being self-modified. Two distinct regions can be identified in a ribozyme: the binding region which gives the ribozyme its specificity through hybridization to a specific nucleic acid sequence (and possibly also to specific proteins), and a catalytic region which gives the ribozyme the activity of cleavage, ligation or splicing.
It has recently been proposed to use ribozymes in order to treat diseases or genetic disorders by cleaving a target RNA, such as viral RNA or messenger RNA transcribed from genes that should be turned off. This was proposed as an alternative to blockage of the RNA transcript by the use of antisense sequences. Owing to the catalytic nature of the ribozyme, a single ribozyme molecule cleaves many molecules of target RNA and therefore therapeutic activity is achieved in relatively lower concentrations than those required in an antisense treatment (WO 96/23569).
The use of ribozymes for diagnostic purposes has been only seldomly mentioned. WO 94/13833 describes a method for detecting nucleic acid molecules in a solution by tailoring a specific ribozyme molecule having two regions, one complementary to the nucleic acid sequence to be detected, and the other complementary to a co-target molecule bearing a detectable label. The ribozyme is able to specifically and reversibly bind both to a selected target nucleic acid sequence and to the labeled co-target. When both the target and the co-target are bound, the ribozyme undergoes a conformational change which renders it active and able to cleave the label of the co-target, and the free label can then be detected. Upon cleavage of the co-target, the ribozyme is able to re-associate with an additional co-target, cleaving more label and producing more detectable signals.
WO 94/13791 concerns a regulatable ribozyme molecule which upon binding to a ligand alters its activity on a target RNA sequence. Again, as in WO 94/13833, binding to the target causes a conformational change in the ribozymes which renders it active. An example for such a change, is in the presence in the ribozyme of a redundant sequence which masks the core region of the ribozyme. Only upon binding of the target sequence to said redundant sequence, the core becomes unmasked, and thus active.
U.S. Pat. No. 5,472,840 (Stefano, J. E.) discloses a nucleic acid sequence comprising the MDV-1 motif (capable of autocatalytic replication in the presence of the enzyme of the Q-beta replicase) which becomes active only when it forms a specific structure, comprising the sequence GAAA, with a target nucleic acid molecule. The ribozyme of Stefano is prepared by modifying an existing ribozyme and features only the activity of cleavage in the presence of a very restricted range of target molecules.
Lizardi, M. P. and Helena Porta (Biotechnology 13:161-164 (1995)) disclose an allosteric hammerhead ribozyme that is a priori inactive, and is triggered by specific interaction with a DNA allosteric effector that is complementary to a single-stranded loop in the RNA stranded ribozyme. This publication discloses a ribozyme which becomes active when a part of which becomes double-stranded.
The above publications disclose ribozymes which become catalytically active, either due to a conformational change or due to a hybridization reaction which render them double-stranded. These types of reactions may also occur spontaneously, for example, if a ribozyme is inactive due to the presence of a redundant sequence which masks its core region, this redundant sequence may either break, or open, even in the absence of the target, and thus the ribozyme will become catalytically active even without a target. Spontaneous reversion to an active state, of course renders the ribozymes impractical for diagnostic purposes.
Israel Patent Applications 112799 and 115772 (corresponding to PCT/US96/02380) disclose methods for the detection of catalytically active ribozymes in a medium, wherein typically the catalytically active ribozyme is used as a reporter for the presence of other biomolecules in a test sample. In accordance with the methods disclosed in these applications, catalytically active ribozyme, if present in an assayed medium, yields a reaction cascade in which more catalytically active ribozymes are produced in a positive-feedback manner in one of various embodiments specified in these applications. The catalytically active ribozyme may be produced, or activated only in the presence of an assayed molecule.
In the following, the term xe2x80x9cnucleozymexe2x80x9d will be used to denote an oligonucleotide or a complex formed between an oligonucleotide and a nucleic acid sequence or between an oligonucleotide and another molecule e.g. an oligonucleotide, a protein or a polypeptide, etc., which possesses a catalytic activity. An example of a nucleozyme is a ribozyme.
The term xe2x80x9cproto-nucleozymexe2x80x9d will be used to denote a nucleic acid molecule or a complex of two or more such molecules, which has a priori no catalytic activity but which becomes catalytically active upon formation of a complex with a co-factor. A proto-nucleozyme is in fact a nucleozyme with a missing component, which missing component is completed by the co-factor. The complex between the proto-nucleozyme and the co-factor may also at times be referred to as xe2x80x9ccatalytic complexxe2x80x9d and is in fact a nucleozyme since it has catalytic activity. The proto-nucleozyme may consist of deoxynucleotides (dNTP""s), ribo-nucleotides (rNTP""s), as well as other nucleotides such as 2xe2x80x2-O-methyl nucleotides, or any combinations of these.
The term xe2x80x9ccatalytic activityxe2x80x9d is meant to encompass all possible catalytic activities of nucleozymes, including cleavage, ligation, splicing-out (cleaving both ends of a short nucleic acid sequence to remove it from a longer sequence and ligating the ends of the cut), splicing-in (cleaving open a nucleic acid sequence, inserting another short nucleic acid sequence and ligating the ends of the cut), rearrangement, as well as additional catalytic activities such as phosphorylation, kinase like activity, addition or removal of other chemical moieties, biotinilation, gap filling of missing nucleotides, polymerization, etc.
The term xe2x80x9cco-factorxe2x80x9d will be used to denote a molecule or a moiety within a molecule (e.g. a certain DNA sequence within a larger DNA molecule), which complexes with the proto-nucleozyme to yield a catalytic complex, namely an active nucleozyme. The co-factor completes a missing portion of the proto-nucleozyme so that it can become catalytically active, and turn into a nucleozyme.
The present invention relates to novel proto-nucleozymes and their use. The proto-nucleozymes of the invention have substantially no catalytic activity, as they miss a critical component, which is essential for the catalytic activity, said missing component is completed by the co-factor. In other words, in order to become catalytically active, the proto-nucleozymes need to complex with the co-factor, so as to form a catalytic complex (the nucleozyme) which can exert a catalytic activity. The term xe2x80x9chaving substantially no catalytic activityxe2x80x9d is meant to denote that the proto-nucleozyme possesses either no catalytic activity or possesses a catalytic activity which is very much lower (typically by several orders of magnitude) than that of the catalytic complex.
As pointed out above, there are known ribozymes which complex with a factor (termed also target), e.g. a protein or another nucleic acid sequence which causes the ribozymes to undergo conformational change whereby the catalytic activity of the ribozyme becomes more pronounced. However, in distinction from such prior art ribozymes, the proto-nucleozyme of the invention, a priori misses an essential component and said co-factor provides said missing component. Examples of such missing components are sequences in the core region of the nucleozyme. The co-factor may be a protein which fills a gap between two ends of nucleic acid strands of the nucleozyme or which joins such two ends; or may be a nucleic acid sequence capable of binding between two free ends of the proto-nucleozyme thus filling a gap.
Given the structural an functional features of the proto-nucleozyme, when in solution, it does not have the capability of spontaneously converting into a catalytically active form, since its activation is not dependent solely on a conformational change which may occur spontaneously but rather on completion of a missing component. This feature is a further distinction from prior art inactive ribozymes, which have the capability to spontaneously undergo conformational change, albeit at a low rate, and exert some catalytic activity even in the absence of a co-factor. Thus, unlike prior art inactive ribozymes, such as those described above in the Background of the Invention section of this specification, when the ribozymes of the invention are used, there is substantially no background activity in the absence of the co-factor. For example, when proto-nucleozymes of the invention are used in diagnosis, there is essentially no xe2x80x9cnoisexe2x80x9d, namely, there is a very high signal-to-noise ratio and there are virtually no false-positive results. By another example, when used in therapeutics, the proto-nucleozymes of the invention, will exert a very high target specificity and only in the presence of an appropriate target, the catalytic complex (the nucleozyme) will be formed and exert its activity.
The proto-nucleozymes of the invention can be prepared both by in vitro evolution, in a manner to be described below, or by means of rational design by nucleic acid synthesis, for example, by using a sequence of a known ribozyme and leaving a gap of several missing nucleotides in its core region.
The present invention provides a proto-nucleozyme, which is a nucleic acid molecule or a complex of nucleic acid molecules, which have essentially no catalytic activity but which can complex with a co-factor to form a nucleozyme which possesses a catalytic activity. The proto-nucleozyme lacks a component essential for the catalytic activity of the nucleozyme and said co-factor provides said component.
The component which is missing in the proto-nucleozyme and which is provided by the co-factor for conversion of the proto-nucleozyme into a catalytically active nucleozyme may be a missing nucleic acid segment of one or more nucleotides (hereinafter xe2x80x9cgapxe2x80x9d) or a missing bond between two nucleotides (hereinafter xe2x80x9cnickxe2x80x9d), etc. The co-factor may thus be a nucleic acid segment which can bridge the gap or nick. For this purpose, such a co-factor nucleic acid segment has sequences which can hybridize with complementary sequences in the two strands on both sides of the gap or nick and in the case of a gap, may comprise also the missing sequence. It should be noted that at times the co-factor segment may hybridize to and provide a bridge between two nucleic acid stretches of the proto-nucleozyme other than those immediately flanking the gap or nick. In addition, the co-factor may also at times be a macromolecule such as a protein, an oligosaccharide, etc., which can complex with the two nucleic acid terminals flanking the gap or nick and accordingly bring to bridging of the two. The gap or nick may be between two different oligonucleotide strands which are functionally joined together by said co-factor or may be between ends of the same oligonucleotide wherein the co-factor in such yields the function of a functional closed oligonucleotide loop.
Such proto-nucleozymes may be obtained (xe2x80x9cengineeredxe2x80x9d) by means of in vitro evolution or by means of rational design.
The specific co-factor may be any molecule which can chemically interact with nucleotides in any manner, e.g. by the formation of hydrogen bond, by electrostatic bonds, by Van der Waals"" interactions, etc. Such molecules include, proteins, peptides, oligopeptides, antibiotics, phosphate nucleotides such as ATP, GTT, cyclic AMP, and others, carbohydrates, lipids, nucleic acid sequences (DNA or RNA sequences), etc.
At times it may be desired to modify the proto-nucleozyme in order to increase its nuclease resistance, which may be achieved by replacing some of the natural nucleotides, by non-natural nucleotides such as 2xe2x80x2-O-methyl nucleotides (Usman et al., Nucleic Acids Symposium Series, 31:163-164, 1994).
A currently preferred, but not exclusive, use of the proto-nucleozymes of the invention is as a diagnostic tool for the detection of the presence of a certain agent in a medium. For this purpose, a proto-nucleozyme will be designed such that said agent will be the specific co-factor. When said agent will be present in the tested medium, a catalytic complex will form and the catalytic activity, which will then be assayed, will then serve as a gauge for the presence of said agent in the medium.
By one option the agent may be a disease identifying agent and may be the nucleic acid sequence or an amino acid sequence identifying the disease, for example, identifying an infectious agent or a genetic disease. By another option the agent may be the result of a nucleic acid amplification technique, for example, nucleic acids amplified by PCR, 3SR, NASBA and the like.
For this preferred embodiment, it is preferred to use proto-nucleozymes which possess absolutely no or ummeasurable catalytic activity, and the catalytic activity is essentially manifested then only upon the formation of the catalytic complex. In such case, there will be an essentially zero background signal and a detection of a catalytic activity will then be an unequivocable (xe2x80x9call or nonexe2x80x9d) indication of the presence of said agent (which is the specific co-factor) in the medium. Where a proto-nucleozyme is used which possesses some small catalytic activity, the level of the catalytic activity will have to be determined in order to assay the presence of said agent in the medium.
The present invention further provides a method for the detection of an agent in a medium, which comprises the following steps:
(a) contacting said medium with a proto-nucleozyme, wherein said agent is the specific co-factor required for the formation of a catalytic complex;
(b) providing or maintaining conditions allowing catalytic activity of the catalytic complex; and
(c) assaying for the presence of products of the catalytic activity in the medium, such presence indicating the presence of said agent in the medium.
An example of the type of catalytic activity which may be determined in step (c) above, is cleavage of nucleic acid sequence. For example, the catalytic complex may cleave, from an immobilized nucleic acid substrate, a small fragment bearing a detectable label. Then, detection of a free label in the reaction medium is indicative of the activity of the catalytic complex, which is in turn an indication of the presence of the assayed agent in the medium.
Other examples of catalytic activity which can be determined in step (c) are ligation, splicing out, splicing in, etc. All the above three catalytic activities result in a change in the distance between two sequences of nucleotides which are the substrates for the reaction. In ligation and splicing out two sequences are brought together and in splicing two sequences are spaced apart. One of these sequences may bear, for example, a fluorescent containing moiety (e.g. rhodamine), and the other sequence may bear a fluorescent quenching (e.g. fluorescein) containing moiety or a fluorescent enhancing moiety; the change in the distance between the two sequences may then be determined by measuring the change in fluorescence emission, which is quenched (in the case of a fluorescent quencher) or enhanced (in the case of a fluorescent enhancer) when the two sequences are adjacent one another, and enhanced or quenched, res, when the two sequences are spaced apart.
One preferred method for assaying for the products of the catalytic activity is by means of the self amplifying ribozyme cascade reaction disclosed in International Application PCT/US96/02380 and corresponding Israel Patent Applications 112799 and 115772, the contents of which are being incorporated herein by reference. Briefly, the catalytic complex (or the nucleozyme) once formed catalyzes a reaction which brings to activation of a priori inactive nucleozymes (identified in the above-referenced applications as ribozymes), and those catalytically active nucleozymes then act catalytically to activate additional ribozymes and so forth, in a self amplifying positive feedback reaction cascade. This amplified signal thus serves as a gauge for the presence of the initial catalytically active nucleozyme in the medium; the presence of such an initial catalytically active nucleozyme is in turn an indication of the presence of the specific co-factor which is the agent to be detected and which complexes with the proto-nucleozymes to yield the catalytic complex of the initial ribozyme in the medium.
The unique capability of the proto-nucleozymes to become catalytically active in the presence of a specific co-factor, makes them useful also as therapeutic agents in certain targeted therapies. In some diseases, disease-bearing cells differ from normal cells in expression of certain expression products. This is the case, for example, in viral diseases where diseased cells differ from other cells by the fact that they express viral proteins. The proto-nucleozyme may be engineered so that upon complexation with a viral-specific protein, (which will be the specific co-factor), it will have a certain cytotoxic catalytic activity or its catalytic activity will yield a cytotoxic reaction product. The catalytic activity may be targeted at a specific nucleic acid sequence or a gene expression product such that the undesired gene expression (e.g. of a viral origin or of a cellular origin) will be inhibited. For example, the catalytic activity may cleave the sequence of an undesired MRNA, thus inhibiting production of an undesired protein. Such proto-nucleozymes may be provided in formulations allowing their entry into the cell, e.g. a liposome formulation, and while the proto-nucleozymes will enter many cells, they will assume their catalytic activity and will thus destroy only the desired cell population for example, or inhibit the expression of undesired genes only in the viruses bearing cells.
In addition to viral diseases, there are also other diseases where diseased cell express or contain products which are not found in normal cells or found in the latter cells in only small amounts. Such is the case, for example, in cancer; in a variety of infectious diseases other than viral diseases; in various genetic diseases where diseased cells in diseased individuals contain a mutant gene and abnormal expression products; etc.
Accordingly, the present invention provides a method for selectively destroying a specific cell population, containing or expressing a specific agent, the method comprising:
(a) providing a proto-nucleozyme of a nucleozyme, which nucleozyme has a catalytic activity which is cytotoxic to the cell or has cytotoxic reaction products, and wherein said agent is a specific co-factor to said proto-nucleozyme;
(b) inserting said proto-nucleozyme to cells containing or expressing said agent, or applying said proto-nucleozymes to tissue, suspected of containing a population of cells containing or expressing said agent, under conditions or using a vehicle, so as to insert said proto-nucleozyme to said cells.
In the case where the proto-nucleozyme is intended for destroying viral-containing cells, the co-factor may, for example, be a viral-associated protein, e.g. in the case of HIV, the HRV-TAT protein.
The cytotoxicity of the nucleozyme may be manifested in a variety of ways. For example, a nucleozyme, once becoming active, may catalyze a reaction yielding formation of cytotoxic reaction products. Such cytotoxic reaction products may, for example, be reaction production which competitively inhibit one or more essential metabolic or catabolic pathways in the cells. By another example, the catalytic activity of the cytotoxic ribozyme may by itself yield breakdown of substances which are produced within the cells or transported into the cell through the cells"" membranes, which are essential for the cells"" growth and survival. Other examples may be nucleozymes which degrade mRNA, either in general or such having specific sequences, nucleozymes which breakdown tRNAs, nucleozymes which degrade a variety of proteins, and others.
The present invention also provides a method for inhibiting expression of undesired genes in cells, comprising:
(a) providing a proto-nucleozyme of a nucleozyme, which nucleozyme has a catalytic activity which is targeted at a specific nucleic acid sequence or gene expression product so that once active within a cell, the undesired gene expression will be inhibited;
(b) inserting said proto-nucleozyme to cells containing or expressing said gene, or applying said proto-nucleozyme to tissues suspected of having a population of cells containing or expressing said gene, under conditions or using a vehicle so as to insert said proto-nucleozyme to said cells.
By an embodiment of this latter aspect, the co-factor of the proto-nucleozyme is either a nucleic acid sequence of such gene, or a transcription or expression product of the gene.
The inhibition of the DNA expression by this latter method may, for example, by breakdown of the specific mRNA, breakdown of an expression product of the gene, or breaking down regulatory substances regulating expression of the gene.
The above methods where said proto-nucleozyme is inserted into cells, may be useful in human therapy. The proto-nucleozymes within the framework of such therapies may be administered in vivo or may be contacted with cells ex vivo, which cells are then inserted back into the body.
According to its therapeutic aspect, the present invention further provides a pharmaceutical composition e.g. for use in destroying a specific cell population, which comprises said proto-nucleozyme and a pharmaceutically acceptable carrier. The carrier will preferably be of a kind which allows insertion of the proto-nucleozyme into cells, e.g. a liposome carrier or any other carrier known in the art.
As explained above, the nucleozyme of the invention may be produced by rational design or by way of in vitro evolution.
As will be explained further below, in vitro evolution does not require any decision in advance on the exact mechanism of formation of the catalytic complex between the proto-nucleozyme and the specific co-factor. The exact mechanism of activation evolves during such in vitro evolution.
The term xe2x80x9cin vitro evolutionxe2x80x9d, refers to a method of generating and selecting nucleic acid sequences (which may be DNA or RNA sequences, or sequences comprising both dNTP""s and rNTP""s comprising naturally or non-naturally occurring nucleotides) having desired characteristics, without a priori knowing the exact construct of the selected nucleic acid sequence. Typically, it entails production of a huge number of random, or partially random, nucleic acid sequences or complexes comprising one or more nucleic acid sequences, then providing the conditions required for selection of those sequences which feature a specific property (for example, adding a protein and selecting only those nucleic acid sequences which show a catalytic activity only in the presence of the protein). The selected nucleic acid sequences are then amplified, for example, by polymerase chain reaction (PCR), and then selection and amplification steps are repeated over many cycles, e.g. ranging from 10 to 100, resulting in an enrichment of the reaction mixture by those nucleic acid sequences or complexes which feature the desired property. It is at times useful to progressively increase the threshold criteria for selection in each round of a selection and amplification. For example, as the steps of the in vitro evolution proceeds, only species having progressively higher affinity to the desired protein are selected.
In vitro selection methodologies to probe RNA function and structures are summarized in a review by Conrad, R. C., Molecular Diversity 1:69-78 (1995) and have been studied in models for autocatalytic replication of RNA by Giver et al. (G. R. Fleischaker et al. (Editor), Self-Production of Supramolecular Structures 137-146, (1994), Klewer Academic Publishers).
The present invention thus provides an in vitro evolution method for the production of a proto-nucleozyme of the invention which upon formation of a catalytic complex with a specific co-factor manifests a specific catalytic activity which is either absent or considerably lower in the proto-nucleozyme. The method which has both positive and negative selection steps, comprises:
(a) preparing a panel of different nucleic acid molecules, at least part of the sequence in the molecules of the panel is a random or a partially random sequence, such that said part has a different sequence in different molecules of the panel;
(b) adding said co-factor to said panel and incubating under conditions permitting said catalytic activity;
(c) separating between the nucleic acid molecule of said panel which feature said catalytic activity and such which do not, to obtain a first selected panel of nucleic acid molecules which feature said catalytic activity;
(d) amplifying the nucleic acid molecules of said first selected panel;
(e) incubating said first selected panel with a reaction mixture, being devoid of said co-factor, under conditions permitting said catalytic activity;
(f) removing nucleic acid molecules which featured catalytic activity, thereby obtaining a second selected panel of nucleic acid molecules devoid of the removed nucleic acid molecules; and
(g) repeating steps (b)-(f) over a plurality of cycles, e.g. about 10-100 cycles, to obtain said proto-nucleozyme.
By one embodiment, the molecules in the panel are comprised of an entirely random sequence with the exception of two short flanking sequences for attachment of the PCR primers. By another embodiment of the invention the molecules in the panel are constructed based on a known nucleozyme sequence. For example, the molecule may be comprised of a constant, ribozyme-derived sequence attached to a random or semi-random sequence. Generally, a random sequence may be prepared, for example, by utilizing a nucleic acid synthesizer.
In the above method, positive selection steps (steps (b)-(d)) precede the negative selection steps (steps (e)-(f)), this being a preferred embodiment of the invention. However, it is possible also to perform the method in a reverse sequence, namely, first the negative selection steps and then the positive selection steps.
Another example is a panel of molecules, all based, on a known ribozyme sequence, and wherein the nucleotides in the entire molecule or in a part thereof have a certain probability (e.g. 70-99%) of being identical to the known sequence. Such a semi-random sequence means that each nucleotide has a certain probability of being different than the corresponding nucleotide in the original ribozyme (e.g. this probability is 30-1%, respectively). The preparation of the panel begins with a known nucleozyme sequence and then semi-random sequences are created based thereon by replacing some of the nucleotides with the different, random, nucleotides (xe2x80x9cdopingxe2x80x9d). The level of doping is typically about 1-30%. The doping may also be performed in each cycle or once in several cycles, thereby slowly bringing to the evolution of a proto-nucleozyme with a high specificity towards the specific co-factor.
An example of how such selection is made, can be drawn from the specific case where the catalytic activity is cleavaged in cis. Initially, all members of the panel may be immobilized on a solid support. Those sequences which possess the catalytic activity of cis cleavage in the positive selection steps i.e. in the presence of the specific co-factor (steps (b)-(d)) are freed to the reaction mixture and are collected and amplified, for example using PCR. After amplification, the collected sequences are allowed to cleave in the absence of the specific co-factor, and those freed in the negative selection steps (steps (e)-(f)), are removed.
In the steps of selection, it is at times desired to apply the exact conditions in which the proto-nucleozyme will eventually be used. Where the proto-nucleozyme is intended for use within the framework of a diagnostic assay, the conditions of selection will typically be similar to those in the diagnostic assay. For example, where the co-factor is a blood protein, and the proto-nucleozyme is intended for use in a diagnostic assay for detection of such a protein in the blood, the selection (both positive and negative) may be carried out in a reaction medium mimicking the conditions (both the chemical environment and the temperature) which will exist in the assay, e.g. blood-like composition and room temperature.
In the following, a preferred in vitro evolution method, useful for the preparation of proto-nucleozymes of the invention, will be described. It should however be noted that this in vitro evolution method is an example, and the invention is not limited to the preparation of proto-nucleozymes to this specifically described method, neither to in vitro evolution preparation methods in general.
A Preferred in Vitro Evolution Method
A major problem in an in vitro evolution method for the preparation of a proto-nucleozyme having the above defined features, is the difficulty in separating between candidates of proto-nucleozymes which feature catalytic activity only in the presence of the specific co-factor, and those which feature the specific catalytic activity both in the presence of said co-factor as well as in its absence, or in the presence of other agents. In other words, there is difficulty in eliminating the undesired candidates.
Many times, especially in the initial cycles of the in vitro evolution, there are molecules which feature catalytic activity also in the absence of the specific co-factor (which molecules should be removed), but do so at a very low efficiency; thus the negative selection step in which such molecules are removed, may require extremely prolonged incubation times, rendering impractical the whole process of in vitro evolution (which requires multiple cycles of negative selection steps). Under standard incubation times only a small percentage of the molecules which are slow acting but feature a catalytic activity also in the absence of the co-factor, but which should be removed in the negative selection steps, will actually feature the catalytic activity in the given incubation time. This can result in incomplete removal of undesired molecules in the negative selection step, and may eventually lead to false positive results when the proto-nucleozymes are used for diagnosis. In principle, the molecules removed during the negative selection step may be collected, and may be used to xe2x80x9cfish outxe2x80x9d, by hybridization, other identical molecules of the same species of proto-nucleozymes candidates which should have been removed, but due to the short incubation time did not have a chance to feature the catalytic activity. However, the sequences of candidate proto-nucleozymes which feature catalytic activity both in the presence of the specific co-factor and in the presence of other agents, (to be removed), and the sequences of the candidate proto-nucleozymes which feature the desired property only in the presence of said co-factor (to be maintained), may be very similar (the difference in the sequence of the two may be very small relative to the large size of the candidate proto-nucleozyme) so that it would be practically impossible to distinguish between the two by hybridization.
The solution to this problem is the addition of a random tag sequence to each of the candidate proto-nucleozyme in the initial oligonucleotide mixture comprising a panel of different candiproto-nucleozymes, so that after amplification, all oligonucleotides of the same species (i.e. all molecules amplified from an original parent molecule) have the same random tag sequence. This tag sequence, does not form a part of the functional sequence of the nucleozyme (i.e. the tag is a redundant sequence). The functional sequence is the part that when the proto-nucleozyme reacts with the co-factor which is the part which imparts the catalytic activity. The tag sequence is linked to a variable sequence (candidate for evolving to the functional sequence). While the variable sequence of different oligonucleotide species may be similar (for example since all variable sequences were xe2x80x9cdopedxe2x80x9d from an original known sequence of a ribozyme); the tag sequence is completely different from one oligonucleotide species to the other.
Thus, when, following a negative selection step, oligonucleotides featuring catalytic activity in the absence of said co-factor are collected in order to be removed, it is then possible to first cleave the cleavable sequence, and thus to collect separately only the tag sequences of the undesired oligonucleotides. These tag sequences, which are random, have a very high probability of being different in each species of oligonucleotide molecules, and thus may be used to effectively xe2x80x9cfish outxe2x80x9d complementary tag sequences of other members of the undesired oligonucleotide species to be removed and are thus capable of discovering xe2x80x9clatentxe2x80x9d oligonucleotides which, if the incubation time was long enough, would have featured catalytic activity even in the absence of the co-factor. Since the xe2x80x9cfishing outxe2x80x9d i.e. elimination process is based only on the hybridization of the unique tag sequence (which is different than the tag sequences of the other species), the fact that the variable sequence of the oligonucleotides which are to be removed is very similar to the variable sequence of the oligonucleotides which should be maintained, does not interfere with the selection process.
Thus, the in vitro evolution method for obtaining proto-nucleozyme of the invention, which together with a co-factor form a catalytic complex having a catalytic activity imparted by a functional sequence of said proto-nucleozyme, comprises:
(a) preparing a mixture of different oligonucleotide candidates for evolving to said proto-nucleozymes each of which comprises a variable sequence being a candidate for evolving into said functional sequence and a tag sequence, each of the two sequences being different than corresponding sequences of different oligonucleotides in the mixture, the variable sequence and the tag sequence being linked by at least one cleavable sequence;
(b) processing the mixture through positive and negative selection steps, each step optionally followed by an oligonucleotide amplification step, there being at least one positive selection step and at least one negative selection step, these steps comprising:
(ba) a positive selection step comprising applying said specific co-factor and separating. between the oligonucleotides which have and those which do not have catalytic activity to obtain a first selected mixture comprising a first group of oligonucleotides featuring catalytic activity in the presence of the co-factor;
(bb) amplifying said first group of oligonucleotides in said first selected mixture to produce a plurality of copies of each of said first group of oligo-nucleotides to obtain a first amplified mixture;
(bc) a negative selection step comprising:
(bca) applying a reaction mixture devoid of the specific co-factor and separating between a second group of oligonucleotides which do not have catalytic activity in the absence of the co-factor and a third group of oligonucleotides having the catalytic activity in the absence of the co-factor, to obtain a second selected mixture comprising said second group of oligonucleotides and a third selected mixture comprising said third group of oligonucleotides;
(bcb) amplifying said third group of oligonucleotides in said third selected mixture to produce a plurality of copies of each of said third group of oligonucleotides to obtain a second amplified mixture;
(bcc) cleaving the cleavable sequence of the oligonucleotides in the second amplified mixture, separating between the variable and the tag sequences and collecting the tag sequences;
(bcd) contacting the collected tag sequences with the second selected mixture under stringent conditions of hybridization and removing hybridized oligonucleotides from other oligonucleotides of the second mixture, thereby obtaining a fourth selected mixture of oligonucleotides essentially devoid of oligonucleotides which have catalytic activity in the absence of the co-factor;
xe2x80x83where said positive selection step precedes said negative selection step, said positive selection step is applied on said mixture prepared in step (a) and said negative selection step is applied on said first amplified mixture; and where said negative selection step precedes said positive selection step, said negative selection step is applied on said mixture obtained in step (a) and said positive selection step is applied on said fourth mixture.
Preferably, the positive selection step should precede the negative selection step. It is preferable that in the positive selection step of each consecutive cycles the conditions become more and more stringent, for example, shorter assay times, harsher sample preparation conditions etc.; while in the negative selection step conditions become less and less stringent, allowing even those oligonucleotides with a very small catalytic activity in the absence of the co-factor, to exert their catalytic activity (and thus to be removed) for example by utilizing longer incubation times, etc.
Where the negative selection step is repeated a plurality of times, each step or several steps may be performed in the presence of another agent which is not the co-factor. For example, where the co-factor is a certain protein, each step in the negative selection may be incubated in the presence in the medium of another non-co-factor protein being present in a different negative selection step.
It should be noted that by the above in vitro evolution method, proto-nucleozymes which are converted into nucleozymes by mere conformational change are eliminated. Such proto-nucleozymes can convert into a catalytically active form (i.e. into the nucleozyme) even spontaneously, without the co-factor, albeit at a low probability. Due to the unique and highly sensitive negative selection step employed in the above in vitro evolution method, all such proto-nucleozymes are identified and are removed, retaining only those proto-nucleozymes which have essentially no catalytic activity and need the co-factor, which complete the missing component, to become catalytically active.
The present invention will now be illustrated in the following non-limiting examples with an occasional reference to the annexed drawings: